Mechanosensitive ATP release from hemichannels and Ca2+ influx through TRPC6 accelerate wound closure in keratinocytes

Abstract

Cutaneous wound healing is accelerated by exogenous mechanical forces and is impaired in TRPC6 knockout mice. Therefore, we designed experiments to determine how mechanical force and TRPC6 channels contribute to wound healing using HaCaT keratinocytes. HaCaT cells were pretreated with hyperforin, a major component of a traditional herbal medicine for wound healing and also TRPC6 activator, and cultured in an elastic chamber. At 3 h after scratching on the confluent cells, the ATP release and intracellular Ca2+ increases in response to stretching (20%) were live-imaged. ATP release was observed only from frontier facing the scar. The diffusion of released ATP caused intercellular Ca2+ waves propagating towards the rear cells in a P2Y receptors-dependent way. The Ca2+ response and wound healing were inhibited by ATP diphosphohydrolase apyrase, P2Y antagonist suramin, hemichannel blocker CBX and TRPC6 inhibitor diC8-PIP2. Finally, the hemichannel permeable dye calcein was taken up only by ATP-releasing cells. These results suggest that stretch-accelerated wound closure is due to the ATP release through mechanosensitive hemichannels from the foremost cells and the subsequent Ca2+ waves mediated by P2Y and TRPC6 activation.